Development of effective therapy for acute head injury requires improved understanding of the mechanisms of traumatic brain cell loss. In both clinical and experimental trauma, it is frequently observed that neural damage extends well beyond the limits of the initial physical insult; this delayed propagation of injury suggests the presence of secondary complications which may be amenable to therapeutic intervention. This FIRST proposal is designed to examine the role of cytosolic calcium in neuronal degeneration after trauma, using an in vitro model of mechanical injury to cortical neurons. Preliminary experiments indicate that cell death after neurite transection in cortical cultures is preceded by a sustained failure of neuronal calcium homeostasis. The goal of this project is to characterize the cellular localization and pharmacologic mechanisms of abnormal calcium entry, and to establish the relationships between disruption of neuronal calcium regulation and the subsequent death of the cell. Specific routes of calcium entry to be probed include voltage-gated calcium channels, glutamate receptor-operated channels, sodium-calcium exchange, and leakage through injured neurites. Traumatic neuronal injury will be produced by microknife- or laser-induced transection of a primary neurite, an insult which reliably leads to delayed cell death. Three parameters of cell death will be assessed: (1) cytosolic calcium concentration, using the calcium indicator, fura-2, with digital fluorescence imaging, (2) neuronal morphological changes, using both phase contrast microscopy and the fluorescent membrane label, Dil, and (3) cell survival or death one day after injury. Because brain injury in vivo is often complicated by secondary ischemia, trauma-induced calcium entry will be examined under energy-depleted as well as normal conditions. It is hoped that increased knowledge of the cellular mechanisms of cortical neuronal injury may suggest approaches for effective therapy of traumatic brain injury in vivo.